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Antibiotic
Preparation -
Antibiotics are metabolites having preferential antimicrobial activity. Therefore, they are widely used for curing of human ailments caused by microorganisms. But some of the antibiotics have antitumour activity as well.
The antibiotic penicillin was discovered by Fleming in 1929, but its commercial production could commence only during early 1940s. Although more than 7000 antibiotic compounds have been isolated, only 100 or so are being used to treat human, animal and plant diseases.
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Antibiotic compounds are used either in their natural form or as semisynthetic derivatives the latter are usually produced by isolating the antibiotic nucleus and its chemical modification. Antibiotics are produced by both fungi and bacteria but over 50% of them are obtained from Streptomyces alone.
Inoculum. A high yielding strain is a prerequisite for antibiotic production. Therefore, constant strain improvement is an integral part of commercial production activities. Inoculum development begins on specific solid media, and subsequently specific liquid media are used.
The inoculum is prepared usually in the form of a spore suspension, which is transferred into the fermenters. As a rule, the number of stages between the preserved material and the final inoculum stage is kept to the minimum to minimise the risk of the organism losing its high yield potential.
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Fermenter. Antibiotics are generally produced in stainless steel fermenters (30,000-200,000 1 medium volume) used in the batch or fed batch mode. Agitation is mostly by impellers, but air-lift system is also used. Water cooling is often used to maintain the temperature between 24-26°C for most antibiotic producers.
Generally, the fermenter is maintained at above atmospheric pressure; this reduces contamination risk and enhances O2 supply in the medium. Sterile air is supplied as per need, and for some processes, e.g., penicillin production, materials need to be added throughout the process. In most cases, it is critical to prevent contamination, and suitable cleaning procedures between fermenter runs must be adhered to.
The final stage fermenter is preferably used for antibiotic production for the longest possible period. But the initial stages of fermentation are designed for considerable microbial growth; typically, these are carried out in seed stage fermenters of smaller size.
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One or more seed-stages may be used, depending on the process and the strain, to produce the maximum amount of biomass in the correct physiological state for high antibiotic production when introduced in the final stage fermenter.
Production Medium. Antibiotic production employs a variety of media, a different one for each stage of operation. A considerable research effort is directed at the developing seed-stage and production media to reduce costs and to enhance yields. A typical production medium has about 10% (w/v) solids.
Generally, yields are much higher on complex media. In some cases, a suitable precursor for the antibiotic is also provided, e.g., for penicillin G production phenylacetic acid or phenoxyacetic acid is used as precursor.
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Since the antibiotics are secondary metabolites, the production medium is so designed that a key nutrient becomes limiting at a critical stage to initiate the secondary metabolism in the organism. The nutrient to be made limiting depends on the process, e.g., glucose for penicillin production and phosphate for several antibiotics produced by Streptomyces.
In most production processes, the production fermenter is run in a fed batch mode in which a nutrient, e.g., glucose, is added continuously throughout the fermentation to enhance the duration of antibiotic production. In such a case, small volumes of broth are usually withdrawn to check increase in volume of the broth in the fermenter.
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Antifoaming agents are added at the appropriate stage of fermentation to minimise foaming; silicones, vegetable oils, etc. are used for this purpose. Selected microbiological, physical and chemical features are monitored during the fermentation in order to achieve a proper control on the process.
The nutritional status of broth is monitored regularly to adjust nutrient and precursor (if any) feeding in fed-batch systems. Microprocessors and computers are commonly used to record all the parameters and process changes, and to control various operations like sterilization, etc.
At the end of final production stage incubation, the broth contains only a low concentration (3-35% of the total solutes in the broth) of the antibiotic. The initial step in antibiotic recovery is separation of cells from the broth; this is usually achieved by filtration or centrifugation.
But in some cases, the whole broth is used for extraction. Antibiotic isolation and purification employs solvent extraction, ion exchange, ultrafiltration, reverse osmosis, precipitation and crystallization.
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